This invention relates to cancer diagnostics and therapeutics.
Members of the Myc protein family are involved in the formation of many cancers; in model systems, their heightened expression can induce oncogenic transformation (for reviews, see Cole, Genet 20:361-384, 1986; Luscher and Eisenman, Genes and Development 4:2025-2035, 1990) and apoptosis (Evan et al., 1992; Cell 69:119-128, 1992; Shi et al., Science 257:212-214, 1992), and it can block differentiation (Freytag, Mol. Cell. Biol. 8:1614-1624, 1988; Miner and Wold, Mol. Cell Biol. 11:2842-2851, 1991). Of the Myc proteins, the best studied are probably the c-myc and v-myc products (cMyc and vMyc). These proteins are localized to the nucleus (Dang and Lee, Mol. Cell. Biol. 8:4048-4054, 1988) and activate transcription in transfection experiments when brought to DNA by heterologous DNA binding domains (Lech and Brent, Cell 52:179-184, 1988; Kato et al., Mol. Cell. Biol. 10:5914-5920, 1990; Golemis and Brent, Mol. Cell. Biol. 72:3006-3014, 1992). These proteins contain an activation domain in their amino terminus whose integrity is correlated with Myc's ability to cause oncogenic transformation (Kato et al., 10:5914-5920, 1990; Barrett et al., Mol. Cell. Biol. 12, 3130-3137, 1992. The proteins also contain a conserved structure, the basic region helix loop helix leucine zipper (hHLH-Zip) (reviewed in Vinson and Garcia, The New Biologist 4(4):396-403, 1992), which directs dimerization and DNA recognition (Dang et al., Proc. Natl. Acad. Sci. USA. 89:599-602, 1992; Blackwell et al., Science 250:1149-1151, 1990; Halazonetis and Kandil, Science 255:464-466, 1992). These facts suggest that the biological function of cMyc and vMyc might depend on their ability to bind specific sequences and activate transcription.
This hypothesis has been greatly strengthened by the isolation of Max, a human protein that forms tight heterodimers with cMyc (Blackwood and Eisenman, Science 251:1211-1217, 1991), and the discovery of its murine homolog, Myn (Prendergast et al., Cell 65:395-407, 1991). Max was cloned using an in vitro method that depended on its ability to interact with the cMyc bHLH-Zip (Blackwood and Eisenman, Science 251:1211-1217, 1991). Max protein and mRNA are expressed in all tissues in which cMyc is expressed, and some, including the brain, in which it is not. Two different forms of the protein are encoded from differently spliced transcripts, a 151 amino acid protein (here called Max.sub.1-151 or simply Max) and a larger form (here called Max.sub.1-160) that contains an additional 9 amino acids at the amino terminus of the basic region. Max is localized to the cell nucleus, possibly due to a nuclear localization signal that is present at its carboxy terminus (Kato et al., Genes and Development 6:81-92, 1992). Max has a longer half life than cMyc (&gt;24 h vs 30 min) (Blackwood et al., Genes and Development 6:71-80, 1992; Hann and Eisenman, Mol. Cell. Biol. 4:2486-2497, 1984; Luscher and Eisenman, Mol. Cell. Biol. 8:2504-2512, 1988).
Like Myc proteins, Max contains a bHLH-Zip motif. In Max, this region serves two functions; the helix loop helix and leucine zipper cause Max to form heterodimers with cMyc; and the basic region and residues near it make specific contacts with DNA. Max can form heterodimers with other members of the Myc family (Blackwood and Eisenman, Science 251:1211-1217, 1991) but does not interact with other known bHLH-Zip proteins (Blackwood and Eisenman, Science 251:1211-1217, 1991). Myc/Max heterodimers bind tightly to a consensus CACGTG sequence (Blackwood and Eisenman, Science 251:1211-1217, 1991; Prendergast et al., Cell 65:395-407,1991). Myc/Myc homodimers bind the same sequence less tightly (Blackwell et al., Science 250:1149-1151, 1990; Prendergast and Ziff, Science 251:186-189, 1991; Kerkhoff et al., Proc. Natl. Acad. Sci. USA. 88:4323-4327, 1991; Halazonetis and Kandil, Science 255:464-466, 1992; Papoulas et al., The Journal of Biological Chemistry, 267 15:10470-10480, 1992) presumably because the native protein does not form homodimers readily, so that site recognition occurs only at high protein concentrations. Phosphorylation of Max by casein kinase abolishes DNA binding by Max/Max homodimers but not by cMyc/Max heterodimers, apparently by a direct effect on Max DNA recognition (Berberich and Cole, Genes and Development 6:166-176, 1992).
Because most cMyc in vivo is associated with Max, and because cMyc/Max heterodimers bind the CACGTG site more tightly than cMyc/cMyc homodimers (Blackwood and Eisenman, Science 251:1211-1217, 1991; Prendergast et al., Cell 65:395-407, 1991), it appears likely that one of the functions of Max is to facilitate the binding of cMyc to these sites. It is also possible that association with Max modulates cMyc's gene regulatory function; consistent with this idea, we have recently shown that Max is transcriptionally inert, but that association with Myc greatly potentiates the strength of the cMyc activation function.
These facts have led to a picture of Myc and Max dependent oncogenesis, in which cMyc complexes with Max and binds to sites upstream of genes whose transcription are regulated. In this view, changes in transcription dependent on this complex could be caused by changes in site recognition, by changes in the availability of cMyc or Max, and by modifications to cMyc and Max that alter their ability to oligomerize or that affect their transcription regulatory function. Each of these modulatory steps may be regulated by other cellular proteins, including oncoproteins, which may change the expression or phosphorylation state of the proteins (reviewed in Blackwood et al., Curr. Opin. Genet. Dev. 2:227-235, 1992.